There are two basic methods for creating oligonucleotide arrays. One technique is to synthesize the oligonucleotide array in discrete feature locations on the array, nucleotide by nucleotide, using well-known phosphoramidite synthesis chemistry. This method is called in situ oligonucleotide array synthesis. The phosphoramidites used for in situ synthesis may be spotted on the array using inkjet deposition equipment manufactured by Hewlett-Packard of California, for example, to create a spatially unique pattern of oligonucleotides. Four inkjet nozzles are used to place the different phosphoramidites onto the array substrate. The array of features on the substrate may be physically separate or there may be no space between such features. In situ synthesis involves the repetitive steps of deblocking, coupling, capping and oxidizing, which are well known in the art, until the desired full-length oligonucleotides are synthesized.
While in situ synthesis is a very flexible means for producing DNA arrays, the fidelity or percentage of full-length oligonucleotides synthesized within a feature on the array is less than 100 percent. An ideal array will have only full-length oligonucleotides attached to each feature. The ideal array promotes accuracy in hybridization experiments or assays of target biological materials. If the fidelity of an in situ generated array is less than 100 percent, it typically has non full-length oligonucleotides within a feature that usually consists of shorter lengths of the correct sequence and to a lesser degree, incorrect sequences. Typical DNA coupling efficiencies are around 97 to 99 percent for the standard phosphoramidite chemistry. For oligonucleotides that are 25 nucleotides in length, these efficiencies result in only 46 to 77 percent full-length oligonucleotides contained within a feature (0.97.sup.25 to 0.99.sup.25). This loss of fidelity can cause chemical noise in hybridization experiments and/or difficulty in developing hybridization conditions. The loss of fidelity can also lead to difficulty in interpreting the data.
Photolithography is a method used by Affymetrix in California to produce in situ arrays using procedures that are similar to those used in the semi-conductor industry. In procedure described by Fodor et al. from Affymetrix U.S. Pat. No. 5,405,783, a photo-deprotection step is used where the protecting group on the phosphoramidite is removed by exposing a photosensitive protecting group to light. Four photo masks are used to create patterns to de-protect areas of the substrate and then a nucleotide is added to these regions. This technique requires four masks for each layer of nucleotides. While this technique allows for production of high-density oligonucleotide arrays, it is less efficient than traditional phosphoramidite synthesis chemistry. With efficiencies of about 90 to 95 percent, the percentage of full-length oligonucleotides within a feature is further reduced to about 9 to 27 percent for oligonucleotides that are 25 nucleotides long (0.90.sup.25 and 0.95.sup.25).
Deposition or spotting of pre-synthesized oligonucleotides is another method of creating DNA arrays. The process usually consists of synthesizing the oligonucleotides on a commercial DNA synthesizer, wherein a nucleotide is attached to a solid support at its 3' end and the oligonucleotide is built upon its 5' end using the well known repetitive steps of detritylation, coupling, capping, and oxidation. When the synthesis is complete, a final deprotection and cleavage step is performed to release the 3' end of the first nucleotide from the support for attachment to the array substrate. Before the synthesized oligonucleotide is attached to an array substrate, a purification step is required because the synthesis results in a mixture of full-length product and a percentage of incorrect shorter length oligonucleotides. Without a purification step, the feature fidelity would be similar to the in situ synthesized oligonucleotide arrays, described above.
The purification step can be performed in a number of well-known conventional ways. One method is to use a solid phase column to perform the separation of the full-length sequences from the incorrect shorter length sequences by keeping the trityl-protecting group (DMT) on the last nucleotide in the sequence. This is called "trityl on" synthesis. Only the full-length oligonucleotide sequences should have the trityl group still attached, because shorter length chains have been capped off in the capping step. The solid phase purification column has a high affinity for the trityl group and retains it on the column while allowing the incomplete sequences without the trityl group to pass through. Cleavage of the trityl group from the full-length oligonucleotides is accomplished by applying an acidic solution to the column. Finally, the full-length oligonucleotides are eluted from the column with an acetonitrile and water solution. The eluting solution will contain primarily only full-length product. Another well-known purification method uses liquid chromatography (LC). The synthesized oligonucleotide solution is run on a LC system where the full-length oligonucleotides are separated from the incorrect and short sequences and the fraction containing only the full-length oligonucleotide solution is collected. These purification steps are expensive, time consuming, prone to loss of product, and dilution of the final concentration of the oligonucleotide solution.
At the 3' end attachment to the solid support, typically a linker can be used that contains an amino group, for example. After the oligonucleotide is cleaved from the solid support, the 3'-amino group is available to attach the oligonucleotide to the array substrate. In this conventional procedure, any oligonucleotide synthesized, including the shorter-length or incorrect sequences, will have its 3'-amino group available for attaching to the array surface. Except for the conventional purification step, there is nothing preventing the shorter-length or incorrect sequences from attaching to the array surface during the deposition step. Without a purification step, there is a lower than desired percentage of full-length oligonucleotides in the feature. Thus, to achieve a high-fidelity oligonucleotide feature using the conventional methods, one must purify the solution prior to deposition.
There are two well-known basic techniques for spotting pre-synthesized oligonucleotides onto an array. Pin spotting is one technique where metal pins are dipped into solutions of pre-synthesized oligonucleotides and then touched onto a substrate. A small amount of the solution is transferred to the substrate surface. The other technique uses the inkjet equipment, mentioned above, to spot the solutions of pre-synthesized oligonucleotides. The inkjets are loaded with the pre-synthesized oligonucleotide solutions. The inkjets deposit the oligonucleotides onto the surface in a computer-controlled fashion. Arrays of cDNA are also fabricated using the spotting techniques.
The pre-synthesized oligonucleotides may be linked or attached to the array substrate surface by well-known conventional methods. One method includes the covalent linkage of a chemically modified oligonucleotide (e.g. aliphatic 1.degree. amine) to the substrate surface bearing an amine-reactive group (e.g. aromatic isothiocyanate). Another method includes adsorption to a substrate surface coated with a positively charged polyelectrolyte (e.g. poly-L-lysine), followed by cross-linking to the surface chemically or photochemically (e.g. covalent stabilization via ultraviolet (UV) photo-crosslinking).
Olejnik, J. et al., "Photocleavable biotin phosphoramidite for 5'-end-labeling, affinity purification and phosphorylation of synthetic oligonucleotides", Nucleic Acids Research, 1996, Vol. 24, No. 2, pp. 361-366, describes the synthesis of a photocleavable biotin phosphoramidite (PCB) for attaching to the 5'-end of a synthetic oligonucleotide. The 5'-PCB end of the oligonucleotide binds to streptavidin for streptavidin affinity purification of the oligonucleotide from failure sequences. The PCB is photocleaved after purification. The affinity purification method disclosed by Olejnik et al. is a complex method that includes adding the crude 5'-PCB oligonucleotide to a suspension of streptavidin-agarose beads which are then incubated, spin-filtered, washed and spin-filtered multiple times again, resuspended, irradiated, spin-filtered and washed again.
Thus, it would be advantageous to have a method of making an array of oligonucleotides that comprises essentially only full length oligonucleotides, which did not require the laborious purification steps, and at the same time, would yield a purity of greater than 90% full length oligonucleotides on the array. The arrays so produced could provide higher quality assay results. Such a method would solve a long-standing problem in the art of making arrays.